Activation of Natural Regulatory T Cells by Igg Fc-Derived Peptide 'Tregitopes'

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2008 Activation of natural regulatory T cells by IgG Fc- derived peptide "Tregitopes" Anne S. De Groot University of Rhode Island, [email protected]

Leonard Moise University of Rhode Island, [email protected]

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Citation/Publisher Attribution De Groot, A. S., Moise, L., McMurry, J. A., Wambre, E., Van Overtvelt, L., Moingeon, P., Scott, D. W., & Martin, W. (2015). Activation of natural regulatory T cells by IgG Fc-derived peptide 'Tregitopes'. Blood, 112(8), 3303-3311. Available at: http://www.bloodjournal.org/content/112/8/3303

This Article is brought to you for free and open access by the Institute for Immunology and Informatics (iCubed) at DigitalCommons@URI. It has been accepted for inclusion in Institute for Immunology and Informatics Faculty Publications by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. Authors Anne S. De Groot, Leonard Moise, Julie A. McMurry, Erik Wambre, Laurence Van Overtvelt, Philippe Moingeon, David W. Scott, and William Martin

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IMMUNOBIOLOGY

Activation of natural regulatory T cells by IgG Fc–derived peptide “Tregitopes”

Anne S. De Groot,1,2 Leonard Moise,1 Julie A. McMurry,1 Erik Wambre,3 Laurence Van Overtvelt,3 Philippe Moingeon,3 David W. Scott,4 and William Martin1

1EpiVax, Providence, RI; 2University of Rhode Island, Providence; 3Stallergenes, Anthony, France; and 4University of Maryland, Baltimore

We have identiﬁed at least 2 highly pro- suppression of effector cytokine secre- for the immunosuppressive activity of miscuous major histocompatibility com- tion, reduced proliferation of effector IgG, such as with IVIG, may be related to plex class II T-cell epitopes in the Fc T cells, and caused an increase in cell the activity of regulatory T cells. In this fragment of IgG that are capable of speciﬁ- surface markers associated with TRegs model, regulatory T-cell epitopes in IgG ؉ ؉ cally activating CD4 CD25HiFoxP3 natu- such as FoxP3. In vivo administration of activate a subset of nTRegs that tips the ral regulatory T cells (nTRegs). Coincuba- the murine homologue of the Fc region resulting immune response toward toler- tion of these regulatory T-cell epitopes or Tregitope resulted in suppression of im- ance rather than immunogenicity. (Blood. “Tregitopes” and antigens with periph- mune response to a known immunogen. 2008;112:3303-3311) eral blood mononuclear cells led to a These data suggest that one mechanism Introduction

Induction of specific tolerance to self- or to foreign antigens is the We scanned the Fc region of IgG for natural TReg epitopes that goal of therapy for autoimmunity, transplant rejection, and allergy; may explain (1) tolerance to antibody variable regions and (2) the unresponsiveness is also desirable in the context of therapy with induction of tolerance to selected antigens after administration of potentially immunogenic autologous proteins (such as factor VIII) therapeutic immunoglobulins or Ig fusion proteins.7,8 Using periph- and nonautologous proteins (such as botulinum toxin). Until eral blood mononuclear cells (PBMCs) from individuals allergic to recently, therapeutic tolerance induction relied on broad-spectrum either house dust mite Dermatophagoides pteronyssinus (HDM) or interventions that resulted in widespread effects on immunity, to the major birch tree allergen, Bet v 1141-155, we evaluated the rather than on strategies directed toward restoring a balance effect of these IgG TReg epitopes (“Tregitopes”) in a standard 2-step between effector immune responses and regulatory immune re- “bystander suppression” assay. We explored whether the Tregi- sponses to a specific protein. topes induced aTReg to Bet v 1141-155 using HLA DR*1501 tetramers Natural means of controlling autoimmune responses (natural to the Bet v 1141-155 epitope. We also coadministered HDM lysate tolerance) and of inducing tolerance (adaptive tolerance) are and Tregitopes to HLA transgenic mice and observed suppression known to exist. For example, suppression of inflammation by of immune response to HDM as measured by whole-antibody ϩ ϩ CD4 CD25HiFoxP3 natural regulatory T cells (nTRegs)isan enzyme-linked immunosorbent assay (ELISA) and IL-4 enzyme- important mechanism of effector T-cell regulation, and may linked immunosorbent spot (ELISpot). Further studies need to be represent one of the critical forms of autoregulatory response to performed, but these Tregitopes may provide an explanation for the self-antigens. Upon antigen-specific activation through their TCR, limited immunogenicity of Fc fusion proteins, the expansion of ϩ nTRegs are able to suppress bystander effector T-cell responses to CD4 CD25Hi regulatory T cells after administration of therapeutic unrelated antigens by contact-dependent and -independent mecha- IVIG,8 and the observed effect of immunoglobulin therapy on autoimmune diseases and other medical conditions. nisms. Adaptive TReg (aTReg) induction is one outcome of a T-regulatory immune response, and sustained tolerance (to grafts, to allergens, and to autologous proteins) probably requires the existence of aTRegs with the same antigen specificity as the 1-3 Methods self-reactive T cells. Adaptive TRegs are also known as induced TRegs (iTRegs). However, despite extensive efforts and with few Computational epitope mapping 4,5 exceptions, the antigen specificity of nTRegs is still unknown. To determine whether TReg epitopes exist in immunoglobulin G, we used Natural TRegs may also control immune responses to autologous proteins to which central tolerance may not exist. For example, it the EpiMatrix and ClustiMer epitope-mapping algorithms (EpiVax) to scan the complete amino acid sequence of human IgG sequences derived from has been suggested that T cells need to be rendered tolerant to the the human IgG germ-line heavy and light chain sequences (GenBank variable regions of antibodies that have undergone somatic hyper- accession J00228 and J00241, respectively9). The EpiMatrix system is a 6 mutation. To date, no natural TRegs that respond to IgG epitopes suite of epitope-mapping tools (including EpiMatrix, ClustiMer, and have been identified nor have adaptive TRegs to hypervariable IgG BlastiMer) that has been validated over the course of more than a decade, regions been identified. both in vitro and in vivo (for example, see De Groot et al10 and Koita et

Submitted February 12, 2008; accepted June 14, 2008. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, July 25, 2008; DOI 10.1182/blood-2008-02-138073. payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734. An Inside Blood analysis of this article appears at the front of this issue. © 2008 by The American Society of Hematology

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3304 DE GROOT et al BLOOD, 15 OCTOBER 2008 ⅐ VOLUME 112, NUMBER 8

al11). For this evaluation of IgG sequences, we used EpiMatrix to identify Suppression assays 9-mer peptides likely to bind to at least 1 of 8 common class II alleles HDM lysate. To test for direct (adaptive) suppression of immune response, (DRB1*0101, *0301, *0401, *0701, *0801, *1101, *1301, and *1501).10 sorted and unsorted PBMCs were plated at 5 ϫ 106 Then, using the ClustiMer algorithm, we mapped the EpiMatrix motif cells/well in a 12-well tissue culture dish (Corning, Corning, NY) and stimulated with 2 ␮g/mL matches (for these 8 alleles) along the length of IgG and calculated the D pteronyssinus (HDM) lysate (Greer Labs, Lenoir, NC), HDM lysate with density of motifs for the panel of 8 HLA alleles. Epitope-dense regions, or 10 ␮g/mL Tregitopes, or no peptide and placed in a 37°C incubator with 5% clusters of HLA-binding potential, are usually highly immunogenic in vitro CO and in vivo.12,13 2. After 24 hours, 10 U/mL IL-2 and 20 ng/mL IL-7 (R&D Systems, Minneapolis, MN) were added to each of the wells. The cells were fed every 2 days by half media replacement containing the same concentration of Peptide synthesis cytokines. On day 8, all the PBMCs were collected, washed, and ␮ Peptides were synthesized using solid-phase Fmoc chemistry. Automated restimulated with 2 g/mL HDM lysate. On day 8, all the PBMCs were ␮ ␥ production was carried out on Gilson, Applied Biosystems, and Advanced collected, washed, and restimulated with 2 g/mL HDM lysate in IFN- or Chemtech peptide synthesizers, initially at SynPep (Dublin, CA) and IL-4 ELISpot kits (Mabtech, Nacka Strand, Sweden) according to manufac- subsequently at New England Peptide (Gardner, MA). Tregitope 289 was turer’s instructions. Culture supernatants were analyzed by Searchlight amidated on the C-terminus. Peptides were purified via reverse phase multiplex ELISA assay (Pierce, Woburn, MA). high-performance liquid chromatography (HPLC) to more than 80% purity C3d peptides. Suppression was also tested with a second antigenic and characterized by mass spectrometry. system, C3d. The complement component C3d has recently been estab- lished as an autologous T-helper cell target.15 Human PBMCs were cultured for 8 days as described for HDM lysate with 3 different sets of stimuli: HLA-binding assay 1 control and 2 experimental. The control was a pool of immunogenic C3d Soluble MHC-binding assays were performed as previously described.11,14 peptides alone. The 2 stimulation groups were (1) a pool of immunogenic Nonbiotinylated test peptide was suspended in a 96-well polypropylene plate for C3d peptides with hTregitope 167, and (2) a pool of immunogenic C3d binding measurements at concentrations ranging from 0.1 ␮M to 400 ␮Min peptides with hTregitope 134. Cells were harvested and washed with PBS, triplicate wells. Purified recombinant HLA class II monomers (Benaroya and 2 ϫ 105 cells/well were plated into a 96-well plate and restimulated Research Institute, Seattle, WA), in a solution containing 1 mM PefaBloc, 0.75% with the immunogenic peptide pool alone, the immunogenic peptide pool n-Octyl-B-D-glucopyranoside in 150 mM citrate-phosphate buffer (pH 5.4), and Tregitopes, or no peptide (negative control) for 65 hours. Supernatants were then added to a final concentration of 200 ng/well. The 96-well plates were were analyzed by multiplexed ELISA analysis. Tetramer studies. incubated at 37°C in 5% CO2 for 45 minutes. Biotinylated influenza hemaggluti- PBMCs from 3 different HLA-DRB1*1501 birch nin peptide 307-319 (a known HLA DRB*0101 binder) was added to a final pollen–allergic donors were stimulated weekly with autologous PBMCs ␮ concentration of 0.1 ␮M per well and incubated at 37°C for 20 hours. The pulsed with Bet v 1141-155 peptide (10 g/mL) with or without Tregitopes contents of each well were then added to a 96-well high-binding ELISA plate (10 ␮g/mL). After the third stimulation, cells were stained with the previously coated with the antihuman HLA-DR L243 capture antibody (Becton HLA-DRB1*1501 Bet v 1141-155 tetramer for 45 minutes. The cytokine ϩ Dickinson, San Jose, CA) and incubated at 4°C for 20 hours. The plate was then secretion profile of CD4 Betv1141-155 tetramer–positive cells was subse- developed by addition of 100 ␮L europium-labeled streptavidin at 10 ␮g/mL quently determined using a cytokine surface-capture assay. (Perkin-Elmer, Waltham, MA) and 100 ␮L enhancement buffer (Perkin-Elmer) To establish the phenotypic profile of Bet v 1141-155–specific T cells, to each well. After incubation in the dark at room temperature for 15 to PBMCs from an HLA-DRB1*1501 birch pollen–allergic donor were 30 minutes, fluorescence was measured on a Wallac Victor 3-V time-resolved stimulated weekly with autologous PBMCs pulsed with Bet v 1141-155 fluourometer (Turku, Finland). Binding curves were fitted by nonlinear regres- peptide (10 ␮g/mL) with or without Tregitopes at 10 ␮g/mL. After the sion analysis and concentration that inhibits binding by 50% (IC50) values second stimulation, cells were stained with the HLA-DRB1*1501 Bet v 1141-155 calculated (SigmaPlot, San Jose, CA). Binding strength was categorized based on tetramer and with a combination of antibodies directed against the indicated comparisons with known peptides; an IC50 of 250 ␮M or more is indicative of a surface markers. For control purposes, a subset of cells was also stained weak or nonbinding interaction. with irrelevant tetramers. PBMCs (106) were stained with Fl-labeled antibodies as described16 according to manufacturers’ protocols (eBio- science [San Diego, CA] and BD Biosciences). All stained cells were run on PBMC isolation a FACSCalibur (BD Biosciences), and data were analyzed using FlowJo Peripheral blood samples were obtained from 1 of 3 sources for this study. software (TreeStar, Eugene, OR). In addition, cytokine secretion was Normal donor blood was purchased by EpiVax from the Rhode Island Blood measured by ELISA using PC-conjugated anti–IL-5 and anti–IL-10 antibod- Center in Providence, RI. Citrated peripheral blood was obtained from birch ies (Cytokine Secretion Assay kit from Miltenyi Biotech, Auburn, CA). pollen–allergic patients recruited at Hospital Bećle`re (Clamart, France), after informed consent was obtained according to the Declaration of Helsinki and Coadministration in HLA transgenic mice under the approval of the ComiteĆonsultatif pour la Protection des Patients dans la Recherche Biome´dicale (CCPPRB, Bećle`re). Protocols for blood draws We used HLA-DRB1*0401 transgenic mice produced by Dr Chella David observed US federal and French guidelines and were approved by the respective (Mayo Clinic, Rochester, MN)17 for the studies described here. The lines institutional review boards (CCPPRB and Independent Review Consulting were generated by coinjection of DR alpha and DRB1*0401 beta gene [IRC], Corte Madera, CA). PBMCs were isolated by Ficoll density gradient fragments into mouse embryos to produce knockin transgenes. The DR4 centrifugation, according to the Accuspin protocol (Sigma-Aldrich, St Louis, genes were subsequently introduced individually into class II–negative MO). Cryopreserved PBMCs isolated from dust mite–allergic individuals were H2Abo mice to produce strains expressing class II HLA and no mouse class purchased from Cellular Technologies (Cleveland, OH). II MHC, as determined by FACS analysis. HLA DR4 transgenic mice (4- to 6-week-old females) were injected weekly 3 times subcutaneously (scruff of the neck) with (1) 50 ␮g HDM TReg depletion alone, (2) 50 ␮g HDM and 50 ␮g murine homologue of Tregitope 289, or ϩ To evaluate the role of CD4 CD25Hi cells in the indirect suppression assay, (3) PBS sham control. In a fourth arm, mice were first sensitized to HDM cells were surface stained with anti-CD4 and anti-CD25 fluorescence- through 3 weekly injections of 50 ␮g HDM and then treated with labeled antibodies (BD Biosciences, San Jose, CA) and run on a 3-laser coinjections of HDM (50 ␮g) and Tregitope 289 (50 ␮g). One week after fluorescence-activated cell sorting (FACS) Aria high-speed cell sorter (BD the final injections, mice were killed, and splenocytes harvested and plated Biosciences). CD4ϩ cells with a CD25 mean fluorescence intensity greater in murine IL-4 ELISpot plates as described in “HDM lysate.” The following than 100 were sorted and discarded. Cells from all remaining gates were were added to the plated cells (in triplicate): PBS (no-stimulus control), combined and used in the assay. HDM lysate, purified HDM antigen DerP2, or PHA. From www.bloodjournal.org by guest on October 8, 2015. For personal use only.

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Figure 1. EpiMatrix analysis of human Tregitopes. A EpiMatrix Z scores for eight common HLA alleles are shown, for each of the overlapping 9 mer frames for hTregitope 289 (A) and 167 (B). The EpiMatrix Z score indicates the potential of a 9-mer frame to bind to a given HLA allele; the strength of the score is indicated by the shading. The top 5% of scores are shaded medium and the top 1% of scores are shaded darkest. All scores in the Top 5% (Z-Score Ն 1.64) are considered “Hits.” Scores in the top 10% (shown but not highlighted) are considered elevated; other scores are masked for simplicity. Frames containing four or more alleles scoring above 1.64 are referred to as EpiBars and are highlighted. These frames have an increased likelihood of binding to HLA. Flanking amino acids, added to stabilize the cluster during in vitro B testing, are underlined.

Serum was obtained by cardiac puncture. Quantification of IgG high number of T-cell epitopes compared with expectation based antibody to HDM antigen was determined by antibody-capture ELISA. on similar protein sequences. ␮ HDM antigen DerP2 (10 g/mL) was placed into a 96-well microtiter plate hTregitope 289 and hTregitope 167 were synthesized and overnight at 4°C. The plates were then washed with phosphate-buffered evaluated in soluble HLA-binding assays for their ability to saline containing 0.05% Tween 20 (PBST) and blocked for 3 hours at room temperature with 5% fetal bovine serum (FBS; Gibco, Carlsbad, CA) in compete with high-affinity HLA-specific biotinylated peptides. PBS. Serial dilutions of sera in 0.5% FBS/PBS were added to the plates and IC50 values were calculated by nonlinear regression analysis incubated at room temperature for 2 hours. The microtiter plates were then using the SigmaPlot analysis program. As predicted, the pep- washed with PBST and 100 ␮L goat anti–mouse IgG (gamma-chain– tides bound to multiple HLA molecules with high affinity specific) conjugated to horseradish peroxidase (Southern Biotechnology (Figure 2); Tregitope 167 bound and with high affinity to all 5 Associates, Birmingham, AL) diluted 1:10 000 in 0.5% FBS/PBS was HLA class II alleles, and hTregitope 289 bound with moderate added to each well. Microtiter plates were washed in PBST and then affinity to HLA DRB1*0101 and DRB1*0301, and with high developed with 3,3Ј,5,5Ј-tetramethylbenzidine (TMB; Moss, Pasadena, MD). Absorbances were read at a wavelength of 450 nm measured on a affinity to HLA DRB1*0401 and 0701, but did not bind to HLA Wallac Victor 3-V time-resolved fluourometer. Correction for optical DRB1*1501. imperfections in the plate was made by subtraction of intensities at 540 nm from the 450-nm values. Response to positive control PHA was robust after Activation of nT both immunization conditions and both assay readouts (data not shown). Regs The animal studies protocols were approved by Biomedical Research To determine whether the predicted peptides could activate a subset Models (BRM docket 07-11) and were carried out under Office of of nT , we added the hTregitopes (167 and 289), tetanus toxin Laboratory Animal Welfare assurance number A-4234-01 to BRM. Regs peptide, or solvent control (no peptide) to a culture of freshly isolated human PBMCs in vitro. After 4 days, the cells were Results collected and analyzed by flow cytometry. We found similar increases in CD25 expression in both the tetanus- and hTregitope- Using EpiMatrix and ClustiMer, we analyzed human Fc and stimulated samples, indicating T-cell activation by both peptides defined 2 clusters of MHC-binding motifs. Human Tregitope (Figure 3A; results shown for hTregitope 289). However, expres- ϩ 289 (hTregitope 289, Figure 1A) located in the Fc sequence of sion of FoxP3 within the CD4 CD25Hi subset differed significantly IgG, contains 2 frames with EpiBars, features characteristic of depending on the stimulus used. Tetanus stimulation led to an promiscuous epitopes. Interestingly, the predicted epitope is expected decrease in expression of FoxP3, suggesting that the ϩ located in the region known to be involved in FcR binding and is CD4 CD25Hi subset was primarily T effector cells (Teffs), whereas conserved in all IgG allotypes but is not conserved between Tregitope stimulation led to a more than 2-fold increase in isotypes. Human Tregitope 167 (hTregitope 167, Figure 1B) expression of FoxP3, suggesting activation of nTRegs (Figure 3B), does not contain a classic EpiBar; however, it does contain a thereby confirming the expected activity of the Tregitopes.

DRB1 DRB1 DRB1 DRB1 DRB1 ␮ ID AA Sequence *0101 *0301 *0401 *0701 *1501 Figure 2. HLA binding results. The IC50 value ( M) and afﬁnity interpretation for each of the epitopes is shown. Based hTregitope 289 EEQYNSTYRVVSVLTVLHQDW Moderate Moderate high high non- binder on comparisons with known peptides IC50 scores lower than 25 ␮M indicate high binding, while an IC above 400 ␮Mis hTregitope 167 PAVLQSSGLYSLSSVVTVPSSSLGTQ high high high high 50 high indicative of a weak or nonbinding interaction. From www.bloodjournal.org by guest on October 8, 2015. For personal use only.

3306 DE GROOT et al BLOOD, 15 OCTOBER 2008 ⅐ VOLUME 112, NUMBER 8

AB TReg depletion experiment. As shown in the previous 2 assays, 4 costimulation of PBMCs with HDM lysate and hTregitope leads to 10 4 a signiﬁcant reduction of effector cytokine secretion in a bystander 3 10% 10 3 suppression assay. Depleting CD4ϩCD25 cells by ﬂow sorting 13% Hi 102 2 before the coincubation step results in a reduction in the amount of ϩ bystander suppression, indicating that CD4 CD25Hi cells (TRegs) 101 1 are essential for Tregitope-mediated suppression. (Figures 6A,B).

TetTox Peptide TetTox 0 10 0 Phenotypic markers of aTReg expansion. To further character- ϩ ce 104 4 ize modulation of cytokine responses by allergen-speciﬁc CD4 54% T cells after hTregitope costimulation, we investigated the pheno- 3 9% 10 3 type of the cells responding to coincubated antigens by combining 102 2 MHC class II tetramer staining with a cytokine surface capture assay. PBMCs were isolated from 3 individuals with severe birch

101 1 D25 fluorescen D25

Tregitope tree allergies and stimulated with an HLA DR1*1501–restricted C 100 0 immunodominant peptide (aa’s 1141-1155) from the major birch 4 4 10 # CD4+CD25+ gated cells tree allergen, Bet v 1. Representative results are shown in Figure 7; results are also summarized in Table 1. 103 6% 3 20% To understand the effect of Tregitope costimulation in allergy, 102 2 ϩ we separated Bet v 1141-155–speciﬁc CD4 T cells into 2 popula- 101 1 tions, one stimulated with Bet v 1141-155 peptide alone and one

No Peptide costimulated with Bet v 1141-155 peptide and hTregitopes 167 and 100 0 100 101 102 103 104 100 101 102 103 104 289 (coincubation with Tregitope 289 induced similar modification of the cell surface markers; data are available for only one subject, CD4 fluorescence FoxP3 fluorescence of CD4+ CD25+ gated cells not shown). Cell surface flow cytometric analysis of each popula- Figure 3. Activation of natural Tregs in the presence of Tregitope. Human PBMC tion was performed after 2 weeks of culture. As shown in Figure ϩ were stimulated directly in vitro for 4 days in the presence of tetanus toxin peptide 7A, most Bet v 1141-155–specific CD4 T cells were contained (TT830-844), Tregitope, or no stimulus. A. Cells were stained extracellularly with within the CD45ROϩCD25ϩC62LϪCCR7Ϫ (activated effector anti-CD4 and anti-CD25 and intracellularly with FoxP3 and analyzed by flow cytometry. Incubation with Tregitope increased the percentage of CD4ϩCD25ϩFoxP3ϩ memory) T-cell population. After coincubation with hTregitope ϩ T cells (54%) compared to TT830-844 (13%) or no stimulus (20%). Numbers in boxes 167, a slight increase of tetramer-stained CTLA-4 T cells was are the percentage of cells in that gate. B. From top to bottom, FoxP3 expression is observed (Figure 7A), suggesting a possible conversion from Th2 shown in PBMC from the same subject following incubation with Tetanus toxin to aT in this mixed population of tetramer-stained cells. For the epitope (13%); hTregitope 289 (54%) and no stimulus (20%). Regs 2 subjects (donor 1 and donor 2) who had detailed cell surface marker phenotypes performed, an increase in CTLA-4 expression

Induction of aTRegs of 3- and 5-fold was observed (from 6.2% to 19.7%, and from 6.6% ϩ to 33.8% of Bet v 1141-155 CD4 T cells, respectively, in the In nonallergic individuals, aTRegs suppress immune responses that presence of hTregitope 167, compared with stimulation performed lead to the generation of IL-5 and allergen-specific IgE. We with Bet v 1141-155 peptide alone). Tregitope coincubation led to a therefore evaluated whether coincubation of hTregitopes and HDM relative increase in CCR7-expressing cells, suggesting that the ϩ 18 lysate could suppress cytokine secretion in PBMCs from HDM- Betv1141-155–specific CD4 TRegs were central memory TRegs. allergic individuals. PBMCs from HDM-allergic individuals were Tregitope coincubation also led to a significant decrease in stimulated with HDM lysate with or without Tregitopes for 8 days, expression of CCR4, CD30, CRTH2, and CCR6, which have been washed, and restimulated with HDM lysate alone. Supernatants shown to be associated with Th2 responses.19,20 ϩ were collected on day 10 and cytokine secretion was analyzed by Most Bet v 1141-155–specific CD4 T cells from birch pollen– ELISA (Figure 4). Expansion of aTRegs was measured by FACS. allergic donors are “Th2-committed” and produce high levels of IL-5 As shown in Figure 4, coincubation of PBMCs with DM after Bet v 1141-155 stimulation, as shown in Figure 7B. In contrast, IL-5 ϩ ϩ antigen and Tregitopes leads to expansion of CD4 CD25Hi cells; secretion was significantly reduced when Bet v 1141-155–specific CD4 increases of 3.7-fold and 4.7-fold (over DM antigen alone) were T cells were coincubated with Bet v 1141-155 peptide and hTregitope 167 observed after hTregitope 167 coincubation and hTregitope 289 and hTregitope 289 (Table 1). coincubation, respectively. Suppression of immune response to a coincubated antigen, in the presence of an expanded population of Dust Mite Dust Mite + Dust Mite+ ϩ CD4 CD25Hi T regulatory cells, suggests that aTRegs were induced alone hTregitope 289 hTregitope 167 during the coincubation. 104 104 104 3 3 3 7.48 Furthermore, as shown in Figure 5, coincubation of PBMCs 10 1.6110 5.87 10 with a pool of immunogenic peptides derived from C3d and 102 102 102 hTregitopes leads to suppression of IFN-␥, IL-5, and IL-6 (T effec- 101 101 101 tor cytokines) and induction of IL-10 and MCP-1 (cytokines 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 associated with TReg function) compared with antigen alone. Thus we have documented the following for both hTregitope 289 and CD25 Fluorescence CD4 Fluorescence ϩ Hi hTregitope 167: (1) the expansion of antigen-specific CD4 CD25Hi Figure 4. Expansion of TReg (CD4/CD25 cells) following coincubation with Tregitope. The expansion of CD4ϩCD25Hi T cells among PBMC restimulated with aTRegs after initial incubation with Tregitopes and DM antigen and dust mite lysate following primary incubation of PBMC with either dust mite lysate (2) associated up-regulation of TReg cytokine secretion and down- alone or dust mite lysate and hTregitope 289, or dust mite lysate and hTregitope 167. regulation of T effector cytokine secretion. FoxP3 was not assessed. Numbers in boxes are the percentage of cells in that gate. From www.bloodjournal.org by guest on October 8, 2015. For personal use only.

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Figure 5. Response to immunogenic peptides in the 3.5 presence of Tregitope 167. Responses to antigen Response following incubation with C3d alone Response following incubation with C3d + Tregitope 134 restimulation following initial stimulation with a pool of 3.0 immunogenic peptides derived from C3d protein (f); Response following incubation with C3d + Tregitope 167 C3d peptides plus Tregitope 134 ( ); and C3d peptides 2.5 plus Tregitope 167 ( ). Responses are shown as fold increase over background, which was no stimulus (con- 2.0 trol) in the secondary incubation. The respective base- line (background) values in pg/ml are indicated within the 1.5 x-axis labels. There was no difference in levels of IL-4, ␣ ␤ TNF , or TGF 1 (data not shown). 1.0 0.5 - (Background = no restimulus) Fold Increase over Background hIFNg hIL5 hIL6 hMIP1a hIL10 hMCP1 background: background: background: background: background: background: 1.78 pg/mL 1.94 pg/mL 1.64 pg/mL 99.08 pg/mL 7.36 pg/mL 298.34 pg/mL

We subsequently evaluated the modulation of cytokine re- The study subjects were selected to be HLA DR1*1501 because ϩ sponses by allergen-specific CD4 T cells after extended Tregitope tetramers with a high affinity for the Bet v 1141-155 epitope were costimulation. After 30 days in culture, Tregitope costimulation available.16 The presence of other class II HLA DR alleles was not contributed to the development of a mixed populations of cells, evaluated in detail. hTregitope 289 did not bind to DRB1*1501 in some of which were no longer IFN-␥ expressing or expressing IL-5 soluble HLA-binding assays. The results of the study suggest that (42% of cells; Figure 7C bottom left quadrant), and some of which the effect of hTregitope 289 was diminished in these HLA (44%) demonstrated a shift from a Th2 to Th1/Th0 response by DRB1*1501 subjects due to failure to bind to at least 1 of their ϩ Betv1141-155–specific CD4 T cells (Figure 7C). This response is 2 HLA class II DRB1 molecules (Table 1). In contrast, hTregitope significant in the context of allergic subjects who have recently 167 binds avidly to HLA DRB1*1501 (IC50 8.4 ␮M; 87% inhibi- been shown to have possible deficits in TReg number and function. tion of binding at 50 ␮M) and was more effective at suppressing Of note, for these HLA DR*1501–positive patients, the effect of IL-5 responses in these subjects. hTregitope 167 was more pronounced (5-fold increase in aTReg induction) relative to the effect of hTregitope 289 (3-fold increase In vivo studies (data not shown). For donor 1, after 10 days in vitro stimulation, compared with Bet v 1141-155 peptide stimulation alone, hTregitope Dust mites cause significant allergic responses in humans, and the 167 costimulation decreased the proportion of IFN-␥–producing mouse model using house dust mite (HDM) lysate is accepted as a CD4 T cells from 4.7% to 1.8% (2.5-fold), and IL-5–producing model that is similar to humans.21 We sought to determine whether the CD4 T cells from 36.4% to 4% (9-fold). Costimulation with Tregitopes could suppress the effector (IL-4 associated) immune re- hTregitope 289 decreased IFN-␥–producing CD4 T cells from sponse to HDM in vivo. As shown in Figure 8, HDM immunization 4.7% to 2.8% (2-fold), and IL-5–producing CD4 T cells from (black bars) provokes a robust response by both IL-4 (Figure 8A) and 36.4% to 6.4% (6-fold). Because the composition of the responding anti-HDM antigen antibody titers (Figure 8B). Both of these responses T cells may be mixed (they were sorted for tetramer staining, not are reduced (gray bars) when HDM lysate is instead coadministered CD4ϩ and CD25Hi), we could observe only a slight increase in with the murine homologue of hTregitope 289. Responses to HDM in IL-10 production, which may reflect aTReg activity. This is also sham-immunized animals were negative as expected. Humoral re- suggested by the change in cell surface phenotype. sponses to HDM antigen (Figure 8B) and IL-4 ELISpot to HDM lysate (Figure 8A) are correlated. Figure 8A shows 38% suppression of ELISpot response in DM naive mice (P ϭ .001) and 84% in the case of A B presensitized mice (P Յ .001). Similarly, ELISA responses to HDM antigen were suppressed by 61.4% (P ϭ .138). Whereas differences as 4 100% 10 measured by ELISpot were highly significant, the difference in antibody 80% 103 response did not reach statistical significance due to a single mouse 60% outlier; were this value to be excluded, the suppression would be 87% 102 ϭ 40% (P .013). This study confirmed the ability of the murine equivalents of

1 human Tregitopes to suppress both antibody and T-cell responses to 10 20%

CD25 fluorescence CD25 coadministered DM antigen in vivo, an effect that is amplified when IFN-gamma secretion 100 0% 0 1 2 3 4 Relative to Dust Mite alone more antigen-specific effector T cells are circulating, as was observed 10 10 10 10 10 Dust Mite Dust Mite Dust Mite Dust Mite alone +Tregitope alone +Tregitope for the HDM-presensitized group of mice. CD4 fluorescence Whole PBMC PBMC depleted of CD4+CD25hi Figure 6. Depletion of CD4؉CD25Hi Treg population reduces suppression of Teff response. Tregitope suppression is dependent on CD4ϩCD25Hi T cells. A. PBMC from allergic individuals were stained with anti-CD4 and anti-CD25 antibodies and Discussion analyzed by flow cytometry. The CD4ϩCD25Hi subset (gate100) was sorted and discarded. B. CD4ϩCD25Hi depleted and nondepleted PBMC were costimulated with Given the many associations among tolerance, the Fc region of HDM lysate with or without hTregitope 289. CD4ϩCD25Hi-depleted PBMC were less ϩ IgG, and the expansion of CD4 CD25Hi T cells after IVIG able to suppress IFN-␥ than were nondepleted PBMC. The levels of IFN-␥ were 8 reduced from 33.5 pg/mL to 11.8 pg/mL in the whole PBMC and from 16.5 pg/mL to therapy, we searched for regulatory T-cell epitopes in the Fc 12.4 pg/mL in the CD4ϩCD25Hi-depleted subset. fragment of IgG. We scanned the complete amino acid sequence of From www.bloodjournal.org by guest on October 8, 2015. For personal use only.

3308 DE GROOT et al BLOOD, 15 OCTOBER 2008 ⅐ VOLUME 112, NUMBER 8

A Figure 7. Birch pollen epitope-speciﬁc immune re- Average of donors 1, 2, and 3 sponses in the presence of Tregitopes. Coculture of PBMC with Bet v 1 allergen with either hTregitope 289 or 100 Bet v 1 alone hTregitope 167 leads to a Th2 to Th1/TReg shift. PBMC Bet v 1 + Tregitope 289 from three birch tree-pollen-allergic subjects were co- Bet v 1 + Tregitope 167 stimulated with Bet v 1141-155 peptide with or without 75 hTregitope. A. Ten to 14 day Tregitope costimulation led ϩ Betv1141-155 tetramer positive CD4 cells to decrease 50 Th2-associated surface markers and to increase regulatory-associated surface markers GITR and CTLA4 (aver-

Percentage of age of donors I, II, and III). B. Coincubation also led to a 25 ϩ decrease of Bet v 1141-155 tetramer–positive CD4 cells secreting IL-5 (left axis) and a modest increase in ϩ Bet-V1 tetramer positive cells 0 Betv1141-155 tetramer–positive CD4 cells secreting IL-10 (right axis). C. PBMC from Donor I were tested under prolonged culture for 30 days; under these conditions, GITR CD25 CD69 CD30 CCR6 CCR7 CCR5 CCR4 CTLA4 CD62L CD62L CRTH2 CRTH2 CXCR3 coculture with Tregitope 167 led to a reduction in the CD45RO B number of Bet v 1141-155–speciﬁc cells secreting IL-5 and to an increase in Bet v 1141-155–speciﬁc cells that were Bet v1 alone Th0 (neither IL-5– nor IFN-␥–secreting, 42%) or Th1 40 Bet v1 + Tregitope 289 2 (IFN-␥–secreting, 44%). Numbers in boxes are the per- Bet v1 + Tregitope 167 centage of cells in the respective quadrant. 30

20 1

Percentage of 10 Percentage of

Bet v1 tetramer positive cells 0 Donor 1 Donor 2 Donor 3 Donor 1 Donor 2 Donor 3 Donor 1 Donor 2 Donor 3 0 Bet v1 tetramer positive cells (day 10) (day 10) (day 14) (day 10) (day 10) (day 14) (day 10) (day 10) (day 14) IL-5 IFN-gamma IL-10 C Bet v1 Bet v1 alone + Tregitope-167 103 3 79% 0% 10 14% 0%

102 16% 5% 102 42% 44%

101 101

IL-5 fluorescence 0 0 (Donor 1: Day 30) 10 10 100 101 102 103 100 101 102 103

Interferon-gamma fluorescence

IgG using the EpiMatrix and ClustiMer epitope-mapping algo- The presence of regulatory T-cell epitopes in the Fc region may rithms. Two strong epitope clusters were identified in the Fc; one explain earlier studies of tolerance demonstrating that immuniza- was found in the CH2 domain of Fc. The hTregitope 289 cluster tion with antigens fused to the IgG or its Fc region could tolerize contained a pattern that we have observed to be common to many against the antigen. Immunoglobulin and the Fc region of IgG have promiscuous and immunodominant epitopes: a characteristic band- been shown to be extremely effective as carriers of therapeutic like pattern representing multiple HLA-binding motifs in a single moieties for nearly 3 decades. The efficacy of these proteins was 9-mer frame. Sequences that contain this bandlike pattern, now originally thought to be due to their long half-life in vivo, as well as termed an “EpiBar,” include tetanus toxin825-850, influenza HA307-319, their ability to bind to Fc receptors. For example, Borel first 22 and GAD65557-567. Interestingly, the predicted hTregitope 289 is located demonstrated that coupling of haptens to murine IgG isotypes led in a region known to be involved in FcR binding, and that is conserved to both T- and B-cell tolerance to the coupled epitopes. Baxevanis in all IgG allotypes but is not conserved between isotypes. et al extended this work by examining the effect of coupling an hTregitope 167, which contains a cluster of putative HLA-binding antigen with human Fc (hFc). They found that hFc could induce motifs, is near the hinge region, and is also highly conserved tolerance in mice, but the truncated Fc domain (CH3) could not among IgG sequences. induce tolerance. They concluded that tolerogenic epitopes may

Table 1. Summary of results for 3 HLA DR1*1501-positive birch pollen–allergic subjects % Suppression of IL-5 response to Bet v 1 Highest-scoring motif match, Sum of EpiMatrix scores 141-155 Tregitope z-score to DRB1*1501 Donor 1 Donor 2 Donor 3 hTregitope 289 2.0 (allele) 2.0 82 59 50 hTregitope 167 2.4 (allele) 5.9 89 86 53 From www.bloodjournal.org by guest on October 8, 2015. For personal use only.

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Figure 8. In vivo studies with mTregitopes. IL-4 and A B antibody responses to HDM lysate and dust mite antigen are shown. Purified HDM antigen DerP2 is a component of HDM P = .0005 P = .138 lysate. HLA DR4 transgenic mice were immunized 3 times 800 P < .0001 with (1) HDM lysate alone, (2) HDM lysate plus Tregitope 289, or (3) sham control. In an additional arm, mice were first 700 0.3 pre-sensitized to HDM lysate by 3 weekly injections followed 600 by 3 additional weekly injections of an equal mixture of HDM lysate plus Tregitope 289. HDM lystate immunization (f) 500 0.2 provokes a robust response by as assessed by IL-4 ELISpot 400 (A) and anti HDM Antigen antibody titers (B). These responses are both significantly reduced (u) when HDM lysate 300 is coadministered with the murine homologue of Tregitope 200 0.1 289. Responses to HDM lysate in sham-immunized animals are negative, as expected. Antibody response (B) and IL-4 100 absorbance at 450 nm per 10^6 background over per 10^6 Not done ELISpot (A) are correlated. Panel A shows 38% suppression 0 0.0 Ͻ of ELISpot response in HDM-naive mice (P .001) and 84% Spot-Forming Cells ELISPOT IL-4 in the case of HDM presensitized mice (P Ͻ .001). Panel B shows 61% suppression of HDM antigen antibody response ELISA (anti-Dust mite antigen antibody) PBS PBS HDM HDM HDM+ ϭ HDM+ HDM alone in HDM-naive mice (P .138); ELISA was not performed for HDM alone Tregitope 289 Tregitope 289 (sham control)

HDM-presensitized mice. Error bars indicate SD. (sham control) pre-sensitization + Tregitope 289 pre-sensitization + Tregitope 289 exist in CH2 domain.23 Zambidis and Scott24 eventually replicated facilitate coexistence with beneficial symbiotic bacteria and vi- these studies, and Phillips et al7 showed that fusion of an IgG heavy ruses. Although we cannot at present directly investigate hTregi- chain to antigen, or administration of the Fc region in conjunction tope 289– and hTregitope 167–specific T cells, we demonstrated with the antigen, could induce tolerance.7,24 Further studies demon- (1) expansion of cells that have the phenotypic and cytokine strated that MHC class II molecules were required for induction of characteristics of nTRegs after incubation (3 day) with the hTregi- tolerance,25 that the IgG carrier region plays an important role in topes; (2) HLA-specific but promiscuous binding; (3) HLA restric- immune suppression by IgG fusion26 but FcR binding is not tion of the suppressive effect of the hTregitopes in coincubation required,25,27 and that CD25 regulatory T cells were required for assays; and (4) abrogation of the suppression after removal of the 28,29 ϩ both the induction and maintenance of tolerance. CD4 CD25Hi cells. Tetramers containing these Tregitopes are in The most recent example of a successful effector-Fc fusion is development; these tetramers will permit the direct enumeration etanercept, a fusion protein between soluble TNF receptor type II and cloning of Tregitope-specific cells for future studies. and the Fc region of human IgG1 that has been exploited for the In addition, we believe that we demonstrated the induction of control of a wide range of chronic inflammatory diseases. Several aTRegs after coincubation of the hTregitopes with several different additional chimeric proteins composed of immunoglobulin con- antigens (HDM, birch pollen). After coincubation for 2 weeks, we stant regions linked to a protein of interest, or fragment thereof, demonstrated the presence of epitope-specific cells that exhibited 30 have been described. These molecules usually possess both the cell surface markers consistent with the induction of aTRegs biologic activity associated with the linked molecule of interest as (CTLA4, GITR). Despite the lack of demonstrated in vitro well as the effector function, and some other desired characteristic hTregitope 289 binding to HLA DRB1*1501, hTregitope 289 also associated with the immunoglobulin constant region (eg, biologic suppressed IL-5 secretion by Bet v 1141-155–stained T cells from stability, cellular secretion). We propose that their efficacy may these HLA DRB1*1501–expressing subjects. This suggested that also be due to the presence of regulatory epitopes in the IgG. the initial stimulation of hTregitope 289–specific nTRegs (restricted Thus, the tolerizing effect of immunoglobulin therapy (such as by the subjects’ other DRB1 allele) was followed by the induction with IVIG), IgG, and Fc could be explained by the occurrence of of HLA DR1*1501–restricted, Bet v 1141-155–specific aTRegs. And natural T epitopes (Tregitopes) in the Fc domain of IgG. We have finally, we have shown that coadministration of the murine version also shown that coadministration of the Tregitopes derived from of human Tregitopes in a murine model of HDM allergy suppresses the Fc region suppresses immune response to an antigen, and that immune response to a coadministered protein in vivo. The suppres- this response is dependent, at least in part, on the presence of sion of immune response to HDM lysate that was observed in vivo regulatory T cells (as defined by the cell surface markers CD4 and has been replicated using other immunogenic proteins (immuno- CD25Hi). In addition, we have shown that the binding and activity genic peptide from the FPX peptibody, botulinum toxin peptides; of these peptides is restricted by HLA, and that the effect is data not shown). We note that Tregitope administration in the independent of Fc binding (these are synthetic peptides and are not context of presensitized mice was more effective. We believe that therefore glycosylated; nor is hTregitope 167 located in the this may be due to the requirement for contact between circulating FcR-binding region). effector T (and B) cells and the nTRegs activated by nTregitope According to current explanations of tolerance, T cells recogniz- administration. At the time of Tregitope treatment, DM lysate– ing self-antigens with high affinity are deleted but autoreactive presensitized mice would have more antigen-specific T cells in the T cells with moderate affinity may escape deletion and be con- circulation, and therefore greater likelihood that an nTReg would 31 verted to function as “natural” regulatory T cells (nTRegs). These encounter an activated T effector after subcutaneous injection of nTRegs are exported to the periphery and suppress autoimmunity. the peptide. We will explore this purported mechanism of action. A second form of tolerance occurs in the periphery where mature T cells are converted to an “adaptive” TReg phenotype upon Proposed model activation via their T-cell receptor in the presence of IL-10 and TGF-␤. The role of these adaptive TRegs (aTRegs) may be to dampen There are at least 2 hTregitopes in human IgG (289 and 167). We effector immune responses (after the primary, vigorous immune have identified at least 6 other putative Tregitopes in IgG (data not reaction, as a means of controlling inflammation), or possibly to shown). We hypothesize that the net effect of positive “effector” From www.bloodjournal.org by guest on October 8, 2015. For personal use only.

3310 DE GROOT et al BLOOD, 15 OCTOBER 2008 ⅐ VOLUME 112, NUMBER 8

Although not directly investigated in our studies, the discovery Regulatory T-epitopes of Tregitopes in IgG points to regulatory T-cell induction after Regulatory IVIG/immunoglobulin therapy, as has been shown in several IgG Processing Presentation T cell 8 36 in MIIC to T cells studies (see for example Ephrem et al and Kessel et al ). It should be noted that there are signiﬁcant differences in the route of administration and the doses given in the referenced murine studies

RegulatoryRegullattory and the subcutaneous doses we administered to dust mite– IgG cytokines sensitized mice. In future studies, the relationship between Tregi-

MHC tope administration (intravenous, subcutaneous) and TReg induction Antigen TCR will be explored using tetramers, and the effect of the route of Presenting Cell administration will be explored. Immunoglobulin therapy has been Effector Effector used in the context of organ transplant and graft-versus-host T cell T-epitopes disease, new-onset type 1 diabetes, multiple sclerosis, and al- in hypervariable region lergy.37-40 Thus, the importance of the discovery of Tregitopes in the Fc region of IgG may extend beyond an explanation of Figure 9. Hypothesized tolerizing mechanism of IgG. We have discovered immunoglobulin-related immunosuppression. The discovery of a conserved T-cell epitopes in IgG that engage natural regulatory T cells. We hypoth- natural regulatory T-cell epitope in the Fc fragment of IgG may esize that antibody-derived Treg epitopes (dark blue epitope) activate regulatory T cells, which leads to suppression of effector T cells that recognize effector epitopes lead to a disease-management paradigm shift, allowing clinicians (red epitope), like those of IgG hypervariable regions to which central tolerance does to safely harness the potential of natural regulatory T cells to not exist. Whether this suppression is mediated by regulatory cytokines alone or by regulate immune responses in a wide range of health conditions. contact-dependent signaling, or both, has yet to be determined. epitopes derived from the antigen and negative regulatory epitopes (Tregitopes) presented on the surface of antigen-presenting cells Acknowledgments (APCs) may influence the overall immune response to the antigen. Fc fragment of IgG (a dimer) contains 2 epitopes that, in the The intellectual contributions of EpiVax collaborator Paul Knopf to absence of associated antigen and danger signals such as Toll-like the development and evolution of the concepts described in this receptor (TLR) ligands, engage cells of a natural regulatory paper are gratefully acknowledged. The following individuals are phenotype. When IgG and antigen (either complexed or separately) recognized for their technical contributions: Christine Malboeuf, are internalized into the same APC (and TLR ligands or other Si-Han Hai, and Daniel Rivera. “danger signals” are present), the response to effector epitopes may outnumber the Tregitopes, resulting in an initial inflammatory response. As antigen is cleared and the ratio of Ig to antigen increases, the balance tips toward tolerance, diminishing immune Authorship response and reducing further tissue damage and adverse systemic effects. (Figure 9). Contribution: A.S.D.G. and W.M. conceived the hypothesis for this This model does not ignore the contribution of Fc receptors to research program, designed the experiments, reviewed the data, IgG-mediated anti-inflammatory processes. Fc-gamma receptors and wrote the paper; J.A.M. provided project management exper- (Fc␥Rs) are required for rapid uptake of IgG and immune tise, reviewed research results in detail, developed all of the figures complexes into antigen-presenting cells during the initial inflamma- for this paper, and assisted with the editing of the paper; L.M. tory phase, and the inhibitory Fc receptor, Fc␥RIIb, increases the assisted with the immunization of the mice and the design and threshold for cell activation during the refractory phase of immune execution of the murine studies; P.M., E.W., and L.V.O. designed response. In our model, Tregitope activation of nTRegs would and performed the birch pollen assays; and D.W.S. provided expert stimulate the release of cytokines such as IL-4 and IL-10 that are guidance regarding previous studies and existing literature, re- known to shift expression from the activating Fc␥RI and Fc␥R IIa viewed the data, suggested additional experiments, and assisted to Fc␥R IIb.32 with the development and editing of the paper. Tregitopes in the Fc region of IgG may also explain how Conflict-of-interest disclosure: Four of the coauthors are employ- antibody variable regions escape immune recognition after they ees of EpiVax (A.S.D.G., J.A.M., L.M., and W.M.) and 2 (A.S.D.G., have undergone somatic hypermutation.33 In addition, as soluble W.M.) are majority stockholders. D.W.S. is a consultant for immune complexes (antibody and antigen) have been shown to EpiVax. E.W., L.V.O., and P.M. are employees of Stallergenes. traverse the placenta34 and tolerance to allergens has been shown to These authors recognize the presence of a potential conflict of be transferable from mother to offspring,35 it invites the question of interest and affirm that the descriptions of experiments represented whether the effect of Tregitopes contained in IgG could be involved in this paper are original and unbiased observations. in the induction of nTRegs and adaptive TRegs in the thymus during Correspondence: Anne S. De Groot, EpiVax, 146 Clifford neonatal development. Street, Providence, RI 02903; e-mail: [email protected]. References

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2008 112: 3303-3311 doi:10.1182/blood-2008-02-138073 originally published online July 25, 2008

Activation of natural regulatory T cells by IgG Fc−derived peptide ''Tregitopes''

Anne S. De Groot, Leonard Moise, Julie A. McMurry, Erik Wambre, Laurence Van Overtvelt, Philippe Moingeon, David W. Scott and William Martin

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